Capillary electrophoresis (CE) has been proposed for rapid fractionation of a variety of biomolecules (Cohen, 1987, 1988, Compton, Kaspar). In the usual CE procedure, the capillary tube is filled with an electrophoresis medium, a small sample volume is drawn into one end of the tube, and an electric field is placed across the tube to draw the sample through the medium. The electrophoretic medium may be a non-flowable polymer or gel material such as agarose gel, but, for many types of separation, may be a flowable fluid medium. Electrophoretic separation of proteins in a fluid electrophoretic medium, based on the differential charge density of the protein species, has been reported (Lauer). For fractionation of nucleic acid species (which have similar charge densities, and therefore must be separated on the basis of size alone) it has been found that high-resolution fractionation can be achieved in a fluid electrophoretic medium containing high molecular weight polymers. This method is described in co-owned U.S. patent application for "Nucleic Acid Fractionation by Counter-Migration Capillary Electrophoresis", Ser. No. 390,631, filed Aug. 7, 1989.
When CE is carried out using a fluid electrophoretic medium, the medium itself may undergo bulk flow migration through the capillary tube toward one of the electrodes. This electroosmotic flow is due to a charge shielding effect produced at the capillary wall interface. In the case of standard fused silica capillary tubes, which carry negatively charged silane groups, the charge shielding produces a cylindrical "shell" of positively charged ions in the electrophoresis medium near the surface wall. This shell, in turn, causes the bulk flow medium to assume the character of a positively charged column of fluid, and migrate toward the cathodic electrode at an electroosmotic flow rate which is dependent on the thickness (Debye length) of the shell.
Electroosmotic flow rate may provide a important variable which can be optimized to improve separation among two or more similar species, as has been described in the above-cited patent application. In particular, when CE is carried out under conditions in which electroosmotic flow occurs in one direction, and the migration of the species to be separated is in an opposite direction, the effective column length for separation for any given species can be made extremely long by making the electroosmotic flow rate in one direction nearly equal to the electrophoretic migration rate of that species in the opposite direction.
Heretofore, attempts to modulate or control electroosmotic flow rate in CE have been limited. In one approach, the pH of the electrophoretic medium is made sufficiently low, e.g., less than pH 2-4, to protonate charged surface groups, and thus reduce surface charge density This approach is not applicable to many proteins where low-pH denaturation effects can occur.
It has also been proposed to include in the electrophoretic buffer, a charged agent which can bind to the surface at a given equilibrium constant, to mask surface charge, and thus reduce electroosmotic flow. This approach is severely limited by the problem of the charged agent binding to the species to be separated, thus altering the charge density and migration characteristics of these species Also, the concentration of binding compound must be calibrated by trial and error.
Attempts to reduce or eliminate electroosmotic flow by covalently derivatizing the charged surface groups in a CE tube with neutral or positively charged agents has also been reported. This approach suffers from the difficulty in calibrating the reaction conditions to achieve a desired electroosmotic flow. In addition, the derivitization reaction is irreversible, i.e., the tube cannot be recoated to achieve other selected electroosmotic flow rates.